Structure and Intrinsic Disorder in Protein Autoinhibition Travis Trudeau, Roy Nassar, Alexander Cumberworth, Eric T.C. Wong, Geoffrey Woollard, Jörg Gsponer  Structure  Volume 21, Issue 3, Pages 332-341 (March 2013) DOI: 10.1016/j.str.2012.12.013 Copyright © 2013 Elsevier Ltd Terms and Conditions

Structure 2013 21, 332-341DOI: (10.1016/j.str.2012.12.013) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 1 Regulation of Autoinhibition In autoinhibited proteins, an inhibitory module (IM, blue) modulates/inhibits the activity of a functional domain (FD, cyan). Equilibrium between active and inhibited states is regulated, that is, autoinhibition can be actively relieved (right side) or reinforced (left side). Reversal of autoinhibition in most systems relies on posttranslational modifications (PTMs), such as phosphorylation, binding to an activating partner, or proteolytic cleavage of the IM (right side). Reinforcement of autoinhibition has been shown to be caused by binding of partners or PTMs (left side). Autoinhibition can be caused by direct, steric interactions of the IM with the FD but can also be induced allosterically. This figure was adapted from Pufall and Graves (2002). See also Tables S2 and S4 and Figure S2. Structure 2013 21, 332-341DOI: (10.1016/j.str.2012.12.013) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 2 Intrinsic Disorder in IMs (A) Box plot of the distribution of disorder in the IM and non-IM regions in proteins from the review of Pufall and Graves (2002) (review data set; n = 39). HP, randomly selected sequences from the human proteome. ∗∗p < 0.001. (B) Box plot of the distribution of disorder for the IMs and FDs of autoinhibited proteins with known structures (structural data set; n = 46). PDB, randomly selected sequences from the PDB. (C) Categorization of the structural set (n = 46) into idIM (green) and sIM (gray). (D) Box plot of the distribution of SASA buried at the IM-FD interface for proteins in the idIM and sIM groups. sP and idP are the SASAs buried by groups of structures and intrinsically disordered proteins, respectively, when in complex with a globular partner (complexes were taken from Mészáros et al., 2007). See also Tables S1–S4 and Figure S1. Structure 2013 21, 332-341DOI: (10.1016/j.str.2012.12.013) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 3 Examples of Autoinhibited Proteins with Known Structure (A–F) FDs and IMs are shown in cyan and blue, respectively. The name or type of the domains in the FD and IM is provided, if available. The dominant mechanism of regulation of autoinhibition is indicated as a symbol in the black squares. The same symbols are used as in Figure 1. Key residues that are important for regulation are highlighted in magenta. (A) Subtilisin BPN’. The protease is autoinhibited by a prodomain. A central event of activation is the proteolytic cleavage of the prodomain. (B) FERM domain of moesin. The domain is inhibited by the C-terminal tail region of moesin. The tail region masks the binding site of the FERM domain. Phosphorylation of a Thr residue is a key step in the activation of moesin. Phosphorylation is believed to promote unfolding of the helices in the tail domain and permit binding of FERM to its partners. (C) Calmodulin-dependent protein kinase I (CaMKI). CaMKI is autoinhibited by an intrinsically disordered segment that contains a calmodulin (CaM) binding region. The IM interacts close to the ATP-binding site, which prevents ATP binding. Binding of CaM to the IM relieves autoinhibition. (D) p47phox. Tandem SH3 domains of p47phox are autoinhibited by the pseudo-substate-like binding of a polybasic IM. Phosphorylation of multiple serines in the IM relieve autoinhibition and enable p47phox to bind target sequences with its SH3 domains. (E) c-Kit receptor tyrosine kinase. The juxtamembrane domain (IM) of c-Kit inserts into the cleft between the N- and C-terminal lobes of the kinase and sterically blocks the conserved kinase motif (DFG) from adopting a productive conformation. Inhibition is relieved via phosphorylation of residues in the IM. (F) Src tyrosine kinase. Phosporylation of a Tyr in the C-terminal tail of the kinase reinforces autoinhibition by promoting intramolecular interactions between the tail and the SH2 domain. In addition, the linker connecting the SH2 domain to the kinase domain adopts a polyproline II conformation that is bound by the SH3 domain. Hence, multiple intramolecular interactions lock c-Src in an allosterically autoinhibited conformation. A key step in the relief of autoinhibition is the dephosphorylation of the Tyr in the C-terminal tail. Structure 2013 21, 332-341DOI: (10.1016/j.str.2012.12.013) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 4 Comparison of idIMs and sIMs (A) Box plot of the distribution of percentages of residues that are phosphorylation sites in idIMs (green) and sIMs (gray). ∗p < 0.05. (B) The numbers of autoinhibited proteins exhibiting (colored) or not exhibiting (white) secondary structural changes upon activation for both idIM and sIM proteins. (C) The numbers of autoinhibited proteins containing (colored) or not containing (white) a splice isoform with altered idIM and sIM. See also Figure S3. Structure 2013 21, 332-341DOI: (10.1016/j.str.2012.12.013) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 5 Disorder Distribution and Sequence Conservation in the idIMs of Homologous Proteins (A–C) Predicted levels of disorder for the FDs (cyan) and IMs (dark blue) of homologous members of the CaM/S100-dependent kinase (A), subtilisin protease (B), and PAK1 kinase (C) families. (D) Box plot of the distribution of sequence entropy per position in FDs, IMs, and among the IIRs of the idIM proteins. ∗∗p < 0.001. See also Figure S5. Structure 2013 21, 332-341DOI: (10.1016/j.str.2012.12.013) Copyright © 2013 Elsevier Ltd Terms and Conditions